PROJECT SUMMARY/ABSTRACT Multiple Sclerosis (MS) is the leading cause of non-traumatic disability in young adults, therapies have limited efficacy and there is no cure. In MS and its mouse model Experimental Autoimmune Encephalomyelitis (EAE), inflammatory T helper (Th)1 and Th17 T cells promote a pathogenic inflammatory neural environment while Th2 and regulatory T cells (Treg) are beneficial. Strategies that inhibit methylation reactions suppress inflammation and Experimental Autoimmune Encephalomyelitis (EAE). These effects have been attributed to inhibition of Protein Arginine Methyl Transferases (PRMT), a family of enzymes that regulate gene expression and activity by catalyzing arginine methylation on histones and other proteins. However, lack of understanding of which and how PRMTs modulate T cell effector function and lack of selective arginine methyltransferase inhibitors has so far prevented further advancement in the field, as well as the clinical application of these findings. We have developed first-in-class PRMT5-specific inhibitors. These inhibitors suppressed EAE and pro-inflammatory Th1/Th17 cell responses while maintaining/increasing Treg/Th2 responses. We hypothesize that PRMT5-mediated symmetric dimethylation reactions contribute to autoimmunity by promoting and sustaining inflammatory T cell responses. Taking advantage of the unique combination of expertise in molecular mechanisms of T cell phenotype and EAE autoimmunity (Guerau), PRMT5-specific inhibitor drugs/signaling pathways (Baiocchi) and novel CRISPRi technology (Han), we propose to identify a mechanism for methylation-promoted autoimmunity by dissecting the role of PRMT5 on specific pathways that drive T cell inflammatory responses. Using both in vitro and preclinical in vivo models of myelin-specific T cell driven MS disease, we propose to: 1) define the signals and regulatory mechanisms governing PRMT5 expression during CD4 T cell activation/differentiation into inflammatory vs. regulatory phenotypes, 2) determine the mechanistic consequences of PRMT5 activity on inflammatory T cell responses and 3) determine the impact of T cell-specific PRMT5 modulation on clinical disease activity in the adoptive transfer EAE mouse model of MS. These experiments will determine the role of PRMT5 in the development and pathogenic potential of myelin-specific T cell responses that lead to CNS autoimmunity. At the completion of these aims, we will have learned how PRMT5-catalyzed arginine methylation shapes the phenotype and expansion of T cells and how these effects shape the adaptive immune response to myelin antigens. These experiments will be the first to address the role of PRMT5 in inflammatory autoimmune T cell responses and disease with novel specific drugs. Since both mouse and human PRMT5 are highly conserved and targeted by these drugs, these studies could translate to novel therapeutic strategies to treat MS and other autoimmune diseases.